Biostar Beta. Not for public use.
ngsplot heatmap scale discrepancy
0
Entering edit mode
12 months ago
Sadia • 50

Hi, I'm using ngsplot to generate a heatmap for ChIP-seq coverage. However, I would like to replot the heatmap myself with the underlying data provided by ngsplot.

I'm a bit confused about the scaling though. The heatmap generated by ngsplot has a scale of (0, 0.9) whereas the max value of the hm data provided by the program goes to 2.5. Is there a subset of data (outliers) that ngsplot ignores during the generation of its plots?

ChIP-Seq • 213 views
ADD COMMENTlink
0
Entering edit mode

which command are you using and did you check the help section/documentation of that function? Most likely, there's some pruning of extreme values so that the dynamic range is not dominated by outliers with very high or very low numbers, but there may also be some z-score normalization or the like going on.

ADD REPLYlink
0
Entering edit mode

To follow up on this, this is what the plotheat function seems to do under the hood:

# Function to plot heatmaps.
plotheat <- functionplot.name, width, height, pointsize, 
    enrichList, title2plot, flood.q=.01){

    # Setup basic parameters.
    ncolor <- 256
    enrich.palette <- colorRampPalette(c('white', 'red'))
    np <- length(enrichList)

    # Filter genes with zero sd. --> this will influence the number of genes that are shown!
    for(i in 1:np){
        g.sd <- apply(enrichList[[i]], 1, sd)
        enrichList[[i]] <- enrichList[[i]][g.sd > 0, ]
    }

  ##... (removed some code)

    # Flooding extreme values which are identified by quantiles. --> this is probably what's 
   # influencing the color scheme the most! Note that flood.q is a variable that can be defined when running plotheat()
    flood.pts <- quantile(c(mat, recursive=T), c(flood.q, 1-flood.q))
    mat[mat < flood.pts[1]] <- flood.pts[1]
    mat[mat > flood.pts[2]] <- flood.pts[2]

##... more code

Code from here. Disclaimer: I've never used that package so there may be additional intricacies.

ADD REPLYlink
0
Entering edit mode

I'm looking at the code, but I'm not able to tell exactly parameter is used as the cutoff limit. Could you shed some light on this?

The function I'm using within the program:

https://github.com/shenlab-sinai/ngsplot/blob/develop/bin/ngs.plot.r

ADD REPLYlink
0
Entering edit mode

what's the command you're typing?

cut-off parameter: flood.q

ADD REPLYlink
1
Entering edit mode
12 months ago
United States

Based on lines 347-358 of ngsplot.r, you should obtain a file "heatmap.RData", which, if loaded via load("heatmap.RData"), should also tell you which flood.frac value was used (default is 0.1 for flood.q and 0.02 for flood.frac as far as I can see)

# Heatmap R data.
if(galaxy==1) {
     heat.dat <- file.path(oname1, 'heatmap.RData')
} else {
     heat.dat <- file.path(oname, 'heatmap.RData')
}
save(reg.list, uniq.reg, ng.list, pts, enrichList, v.low.cutoff, go.algo, 
     ctg.tbl, bam.pair, xticks, flood.frac, hm.color, unit.width, rr, 
     go.list, color.scale, v.lib.size, font.size, go.paras, low.count,
     color.distr, 
     file=heat.dat)
cat("Done\n")
ADD COMMENTlink
0
Entering edit mode

The command I used is the basic one:

ngs.plot.r -G mm9 -R bed -C configuration-file.txt -O outputfile.txt

I loaded heatmap.RData and it does have the parameters. So thank you for that!

Do you know what the difference between flood.q or flood.frac is? My file only contains flood.frac anyways and it is 0.02. So does that translate to 98th percentile of values that are included in the plot?

ADD REPLYlink
0
Entering edit mode

ngs.plot.r -G mm9 -R bed -C configuration-file.txt -O outputfile.txt

I would guess that you can tune that setting in configuration-file.txt

Do you know what the difference between flood.q or flood.frac is

No.

So does that translate to 98th percentile of values that are included in the plot?

This is what the quantile function does:

> quantile(rnorm(100), c(0.02, 1-0.02))
       2%       98% 
-2.075636  2.147820 
ADD REPLYlink
0
Entering edit mode

I've moved the comment about heatmap.RData to an answer since it seemed to help the most. Feel free to challenge that or, otherwise, accept the answer by clicking on the thumbs-up symbol.

ADD REPLYlink
0
Entering edit mode

Thanks for your help!

ADD REPLYlink

Login before adding your answer.

Similar Posts
Loading Similar Posts
Powered by the version 2.1